Liposome loading with metal ions

ABSTRACT

This invention relates to encapsulation of drugs and other agents into liposomes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) of provisionalapplications U.S. Ser. No. 60/326,671 filed 3 Oct. 2001; Ser. No.60/341,529 filed 17 Dec. 2001; Ser. No. 60/356,759 filed 15 Feb. 2002;Ser. No. 60/362,074 filed 7 Mar. 2002 and Ser. No. 60/394,273 filed 9Jul. 2002. The contents of these applications are incorporated herein byreference.

TECHNICAL FIELD

This invention relates to encapsulation of drugs and other agents intoliposomes.

BACKGROUND OF THE INVENTION

Liposomes are microscopic particles that are made up of one or morelipid bilayers enclosing an internal compartment. Liposomes can becategorized into multilamellar vesicles, multivesicular liposomes,unilamellar vesicles and giant liposomes. Multilamellar liposomes (alsoknown as multilamellar vesicles or “MLV”) contain multiple concentricbilayers within each liposome particle, resembling the “layers of anonion”. Multivesicular liposomes consist of lipid membranes enclosingmultiple non-concentric aqueous chambers. Liposomes that enclose asingle internal aqueous compartment include small unilamellar vesicles(SUVs) and large unilamellar vesicles (LUVs). LUVs and SUVs range insize from about 50 to 500 nm and 20 to 50 nm respectively. Giantliposomes typically range in size from 5000 nm to 50,000 nm and are usedmainly for studying mechanochemical and interactive features of lipidbilayer vesicles in vitro (Needham et al., Colloids and Surfaces B:Biointerfaces (2000) 18: 183-195).

Liposomes have been widely studied and used as carriers for a variety ofagents such as drugs, cosmetics, diagnostic reagents, and geneticmaterial. Since liposomes consist of non-toxic lipids, they generallyhave low toxicity and therefore are useful in a variety ofpharmaceutical applications. In particular, liposomes are useful forincreasing the circulation lifetime of agents that have a shorthalf-life in the bloodstream. Liposome-encapsulated drugs often havebiodistributions and toxicities which differ greatly from those of freedrug. For specific in vivo delivery, the sizes, charges and surfaceproperties of these carriers can be changed by varying the preparationmethods and by tailoring the lipid makeup of the carrier. For instance,liposomes may be made to release a drug more quickly by decreasing theacyl chain length of a lipid making up the carrier.

Liposomes containing metal ions encapsulated in the interior of thevesicle have been used in diagnostic applications. For example,liposomes have been used for delivery of contrast agents with the goalof accumulating a contrast agent at a desired site within the body of asubject. In the latter application, liposomes have mainly been used fordelivery of diagnostic radionucleotides and paramagnetic metal ions ingamma and magnetic resonance imaging, respectively. This includesliposomal encapsulation of radionucleotides such as ¹¹¹In, ^(99m)Tc and⁶⁷Ga and paramagnetic ions such as Gd, Mn and manganese oxide. Twomethods are typically employed to prepare liposomes for imagingpurposes. In the first method, the metal is converted to a solublechelate and then introduced into the aqueous interior of a liposome. Inthe second method, a chelating agent derivatized with a lipophilic groupis anchored to the liposome surface during or after liposomepreparation.

Manganese and non-transition metal ions have also been involved inmethods for encapsulation of ionizable agents into liposomes containingan ionophore inserted in the liposome membrane (see U.S. Pat. No.5,837,282 and Fenske et al., Biochim. Biophys. Acta (1998) 1414:188-204). In this method, the ionophore translocates the metal ionacross the liposome membrane in exchange for protons, therebyestablishing a pH gradient. The establishment of an appropriate pHgradient across the liposome bilayer allows the ionizable agent to beencapsulated since the agent can readily cross the liposomal bilayer inthe neutral form and subsequently become encapsulated and trapped withinthe aqueous interior of the liposome due to conversion to the chargedform (see Mayer et al., U.S. Pat. Nos. 6,083,530, 5,616,341, 5,795,589and 5,744,158; Mayer et al., Biochimica et Biophysica Acta (1986)857:123). This work arose from mechanistic studies completed by Deameret al., (Biochimica et Biophysica Acta (1976) 455:269-271) whodemonstrated that liposomes efficiently concentrated severalcatecholamines (dopamine, norepinephrine and epinephrine) in response toa transmembrane pH gradient).

The presence of an acidic liposomal interior and a basic to neutralexterior environment allows agents that are primarily in the neutralform at neutral to basic pH and primarily in the charged form at acidicpH to be readily entrapped within a liposome. Drugs containing ionizablemoieties such as amine groups are readily encapsulated and retained inliposomes containing an acidic interior. This method, where an ionophore(A23187) is used to generate a pH gradient across a manganese-containingliposome, has been used to load topotecan into cholesterol-freeliposomes comprising a PEG-lipid conjugate inserted in the membrane (seeWO/0185131). However, successful loading and retention using atransmembrane pH gradient is realized while the internal pH of theliposome is maintained. Since the pH gradient can only be maintained forshort periods of time, clinical formulation of drugs into liposomesrequires the generation of a pH gradient in liposomes just prior to drugloading. A second disadvantage of this method results from instabilityof lipid, and some drugs, at acidic pH which prevents the need forlong-term storage of the drug loaded liposome. Freezing of liposomalformulations slows the rate of hydrolysis but conventional liposomalformulations often aggregate and leak contents upon thawing unlessappropriately selected cryoprotectants are used.

Complexes between drugs such as doxorubicin or ciprofloxacin anddivalent metal ions such as Mn²⁺ have been reported (Bouma, J., et al.(1986) Pharm. Weekbl. Sci. Edn. 16:109-133; Riley, C. M., et al. (1993)J. Pharm. Biomed. Anal. 11:49-59; and, Fenske, D. B. (1998) Biochim.Biophys. Acta. 1414:188-204). Recently, it was reported that uptake ofdoxorubicin (but not ciprofloxacin) into sphingomyelin/cholesterol LUVscould be carried out with manganese in the internal loading mediumwithout the presence of an ionophore (Cheung et al., Biochimica etBiophysica Acta (1998) 1414:205). It was suggested that a processinvolving both complex formation between doxorubicin and manganese ionsand protonation of doxorubicin inside the liposome resulted in uptake ofthis particular drug in the presence of manganese ions. Stableentrapment of doxorubicin was reported but this work relied on the useof sphingomyelin/cholesterol liposomes, a formulation noted for optimaldrug retention. The methodology reported by Cheung, et al., involvingthe use of MnSO₄ in pH 7.4 HEPES buffer is not reproducible because themetal precipitates from such a buffer.

Various groups have investigated the interaction of metal ions withliposomes with the goal of evaluating the effects of metal cations onvesicle membranes (Steffan et al. (1994) Chem. Phys. Lipids 74(2):141-150). Divalent metal cations such as Ca²⁺ have been implicated inthe unfavourable formation of metal induced crosslinking ofphosphatidylglycerol (PG) containing liposomes due to the negativecharge of the liposome surface. Metal ions have also been implicated inincreasing the phase transition temperature of negatively modeledmembrane systems (Borle, et al., (1985) Chemistry and Physics of Lipids36: 263-283; Jacobson, et al., (1975) Biochemistry 14(1): 152-161).These studies revealed that the addition of calcium todipalmitoylphosphatidylglycerol (DPPG) membranes resulted in a phasetransition temperature increase by about 50° C. These results indicatethat the use of negatively charged lipids in conjunction with metal ionswill result in liposomes that exhibit inferior characteristics for invivo applications.

SUMMARY OF THE INVENTION

This invention is based on the discovery that liposome loadingefficiency and retention properties using metal-based procedures carriedout in the absence of an ionophore in the liposome is surprisinglydependent on the metal employed and the lipid makeup of the liposome. Byselecting lipid makeup and a metal composition, loading or retentionproperties can be tailored to achieve a desired loading or release of aselected agent from a liposome. Furthermore, undesirable precipitationof metal from solutions employed in formulating metal ion encapsulatedliposomes may be avoided by use of metal compatible solutions, andloading may also be enhanced by rigorous removal or complexation ofmetal ions from an external solution containing such liposomes.

This invention thus provides a method of loading an agent into aliposome, comprising preparing a liposome containing an encapsulatedmetal, the liposome being present in an external solution; and, addingto the external solution an agent such that said agent is encapsulatedin the liposome providing that if an agent encapsulated into theliposome is doxorubicin, the encapsulated metal is not solely manganese.In one embodiment of this aspect of the invention, the encapsulatedmetal is a transition metal. Preferably there will be little or no pHdifference between the interior and exterior of the liposome. Morepreferred, the pH will be comparable to the pH of physiological fluidsor an approximately neutral pH. Preferably, the external solution willhave less, more preferably substantially less of the metal. Preferably,the external solution and the surface of the liposomes will beessentially free of the metal in an uncomplexed state. Additionally, thepresent invention provides compositions which are prepared according tothis mhe present invention thus also provides methods for loading agentsinto liposomes, comprising the steps of:

-   -   i) preparing a liposome comprising an encapsulated transition        metal ion and,    -   ii) adding to the external solution of said liposome, an agent        such that said agent is encapsulated in the liposome.        The transition metal ion may be selected from one or more of Fe,        Co, Ni, Cu, Zn, V, Ti, Cr, Rh, Ru, Mo and Pd and may be        encapsulated in a liposome in which Mn is also encapsulated.

The present invention provides compositions which are prepared accordingto this method as well as liposomes containing a transition metal ion ortwo or more different such ions, suitable for use in the method.

The invention also provides a method of loading liposomes using a metalion in a “metal compatible” solution as described herein to minimizeprecipitation of the metal and to maintain it in solution for sufficienttime to prepare the liposome. The present invention thus alsoencompasses a method of loading an agent into a liposome, said methodcomprising the steps of:

-   -   i) preparing a liposome having an encapsulated medium comprising        a metal ion and a metal compatible solution;    -   ii) adding to the external solution of said liposome, an agent        such that said agent is encapsulated in the liposome.        Additionally, the present invention provides compositions which        are prepared according to this method as well as liposomes        containing a metal ion and metal compatible solutions suitable        for use in this method.

Preferably, after drug encapsulation, a liposome of this invention orused in methods of this invention has an extraliposomal pH that issubstantially similar to the intraliposomal pH. More preferably, theextraliposomal and intraliposomal pH is at about pH 3.5 to pH 9.0, morepreferably, it is between about pH 6.0 to pH 8.5, even more preferably,it is between about 6.5 and 8.5, and most preferably, it is betweenabout pH 6.5 and pH 7.5.

This invention is further based on the finding that liposomes preparedto be of low cholesterol content display unexpected loading andretention properties when metal-based loading is utilized. Thus, thepresent invention also provides a method for encapsulating an agent intoa liposome, the liposome being present in an external solution, saidmethod comprising the steps of:

-   -   i) preparing a liposome comprising:        -   a) one or more vesicle forming lipids, providing that the            liposome is of low cholesterol;        -   b) an encapsulated metal in a metal compatible solution;    -   ii) adding to the external solution an active agent such that        the agent is encapsulated into the liposome.

In one embodiment of this aspect of the invention, the metal compatiblesolution includes a transition metal.

In another aspect of the invention, the present invention provides amethod for encapsulating an agent into a liposome, the method comprisingthe steps of:

-   -   i) providing a liposome of this invention in an external        solution, wherein the liposome does not have a transmembrane pH        gradient;    -   ii) adding to the external solution, an agent such that the        agent is encapsulated into the liposome.

Furthermore, this invention also relates to methods of administeringliposomes to a mammal and methods of treating a mammal affected by orsusceptible to or suspected of being affected by a disorder (e.g.cancer). In particular, the invention encompasses a method ofadministering a liposome to a subject comprising administering apharmaceutical composition comprising liposomes of the invention.Methods of treatment or of administration will generally be understoodto comprise administering the pharmaceutical composition at a dosagesufficient to ameliorate said disorder or symptoms thereof. In oneaspect, this invention is based on the finding that liposomes loadedwith active agent using an encapsulated metal display loading andretention properties that are distinct from that displayed by manganese.

This invention provides a liposome composition comprising a liposomecontaining an internal solution comprising one or more encapsulatedtransition metal ions and one or more therapeutic agents, providing thatif the liposome has a lipid composition consisting of sphingomyelin andcholesterol or if the one or more therapeutic agents is solelydoxorubicin, the one or more encapsulated ions is not solely manganese.This invention also provides the aforementioned liposome compositionwherein the liposomes are in an external solution.

This invention also provides a method of loading liposomes with anagent, wherein the liposome composition is a liposome composition asdescribed above, the method comprising: selecting an agent that iscapable of crossing membranes of liposomes in the composition whenpresent in the external solution of the composition but incapable ofcrossing said membranes when in a complex with the one or more metalions in the internal solution, adding the selected agent to the externalsolution of the composition, and maintaining the agent in the externalsolution for sufficient time to load the agent.

This invention also provides methods for preparing, selecting ordesigning liposomes, comprising selecting a metal ion for encapsulationin a liposome to achieve a desired retention of an encapsulated agent inthe liposome. Thus, a method for providing, preparing or selecting aliposome composition having a preferred loading or retention propertyfor a selected agent according to this invention may comprise:

-   -   a) providing a first liposome composition as described above;    -   b) adding the selected agent to the external solution of the        composition of (a) for a time sufficient to provide for loading        of the agent into liposomes of the composition;    -   c) providing a second liposome composition as described above;    -   d) adding the selected agent to the external solution of the        composition of (c) for a time sufficient to provide for loading        of the agent into liposomes of the composition;    -   e) comparing amount of agent loaded or agent retention for        liposomes of the composition resulting at (b) to liposomes of        the composition resulting at (d); and    -   f) selecting, providing, or preparing the liposome composition        resulting at (b) or (d) having a preferred loading or retention,        wherein the liposome composition of (a) and (c) differ by one or        more of: (i) metal ions present in the internal solution; (ii)        lipids in the liposomes of the liposome composition; iii) time        and/or temperature conditions sufficient to provide for loading        of the agents; and iv) the concentration of metal ions present        in the internal solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: A graph showing loading of irinotecan into DSPC/DSPG (80:20mole ratio) liposomes as a function of time using 100 mM Cu(II)gluconatebuffered to pH 7.4 with triethanolamine (TEA) as the internal medium and300 mM sucrose, 20 mM HEPES, 30 mM EDTA (SHE), pH 7.4 as the externalmedium. Loading was carried out at 50° C. at a drug-to-lipid mole ratioof 0.1:1.

FIG. 1B: A graph showing loading of daunorubicin into DSPC/DSPG (90:10mole ratio) liposomes as a function of time using 150 mM CuSO₄, 20 mMhistidine adjusted to pH 7.4 with TEA as the internal medium and SHE, pH7.4 as the external medium. Loading was carried out at 60° C. at adrug-to-lipid weight ratio of 0.1:1.

FIG. 2: A graph showing loading of irinotecan into DPPC/Chol (55:45 moleratio) liposomes as a function of time using 100 mM Cu(II)gluconateadjusted to pH 7.4 with TEA as the internal medium and SHE, pH 7.4 asthe external medium. Loading was carried out at 50° C. at adrug-to-lipid weight ratio of 0.1:1.

FIG. 3: A graph showing loading of epirubicin into DSPC/DSPE-PEG2000(95:5 mole ratio) liposomes as a function of time using 300 mM MnSO₄, 20mM imidazole, pH 7.4 as the internal medium and SHE, pH 7.4 as theexternal medium. Loading was carried out at 60° C. at a drug-to-lipidweight ratio of about 0.2:1.

FIG. 4A: A graph showing loading of irinotecan into floxuridine (FUDR)containing DSPC/DSPG liposomes at an 85:15 mole ratio as a function oftime using 100 mM Cu(II)gluconate, 220 mM TEA, pH 7.4 as the internalmedium and 300 mM sucrose, 20 mM HEPES, pH 7.4 as the external solution.FUDR was passively encapsulated and irinotecan loading was carried outat 50° C. at a drug-to-lipid mole ratio of 0.1:1.

FIG. 4B: A graph showing loading of irinotecan into FUDR-containingDSPC/Chol/DSPG (70:10:20 mole ratio) liposomes as a function of timeusing 100 mM Cu(II)gluconate, 220 mM TEA, pH 7.4 as the internal mediumand either 20 mM HEPES, 150 mM NaCl (HBS), pH 7.4 (•) or 300 mM sucrose,20 mM HEPES, pH 7.4 (∘) as the external buffer. FUDR was passivelyencapsulated and irinotecan loading was carried out at 50° C. at adrug-to-lipid mole ratio of 0.1:1.

FIG. 5: A graph showing loading of irinotecan intocarboplatin-containing DSPC/DSPG (80:20 mole ratio) liposomes as afunction of time using 150 mM CuSO₄ adjusted to pH 7.4 with TEA as theinternal medium and SHE, pH 7.4 as the external buffer. Carboplatin waspassively encapsulated and irinotecan loading was carried out at 60° C.at a drug-to-lipid weight ratio of 0.1:1.

FIG. 6: A graph showing loading of daunorubicin intocisplatin-containing DSPC/Chol (55:45 mole ratio) liposomes as afunction of time using 150 mM CuCl2 adjusted to pH 7.4 with NaOH as theinternal medium and HBS, pH 7.4 as the external medium. Cisplatin waspassively encapsulated and daunorubicin loading was carried out at 60°C. at a drug-to-lipid weight ratio of 0.1:1.

FIG. 7: A graph showing loading of doxorubicin into DPPC/DSPE-PEG2000(95:5 mole ratio) liposomes as a function of time utilizing 300 mM MnSO₄(•) or 300 mM citrate, pH 3.5 (▪) as the internal medium. Doxorubicinloading was carried out at drug-to-lipid weight ratios of 0.1:1 (PanelA), 0.2:1 (Panel B) or 0.3:1 (Panel C) at 37° C. Data points representthe mean of three replicate experiments and the error bars represent thestandard deviation.

FIG. 8A: A graph showing loading of doxorubicin into DMPC/Chol (55:45mole ratio) liposomes as a function of time using 300 mM MnSO₄ (•), 300mM citrate, pH 3.5 (▪) or 300 mM MnCl₂ (▴). Doxorubicin was loaded at adrug-to-lipid weight ratio of 0.2:1 at 60° C. Data points represent themean of three replicate experiments and the error bars represent thestandard deviation.

FIG. 8B: A histogram showing measured transmembrane pH gradients priorto and following doxorubicin loading under various conditions. Thesamples include those based on the citrate loading method prior to(column 1), and after doxorubicin loading (column 2); the MnSO₄ loadingmethod prior to (column 3), and after doxorubicin loading (column 4);and the MnCl₂ loading method prior to (column 5) and after doxorubicinloading (column 6). The results represent the mean pH gradient of threeseparate experiments and the error bars indicate the standard deviation.

FIG. 9: A graph showing loading of irinotecan into DSPC/DSPE-PEG2000(95:5 mole ratio) liposomes utilizing either 300 mM MnSO₄ (O) or 300 mMCuSO₄ (•) as the internal loading medium. Irinotecan was loaded at 60°C. at a drug-to-lipid weight ratio of 0.1:1.

FIG. 10A: A graph showing loading of daunorubicin into DSPC/DSPE-PEG2000(95:5 mole ratio) liposomes as a function of time using 300 mM MnSO₄ asthe internal medium. Loading was carried out at 23° C. (•), 37° C. (∘)and 60° C. (▾) at an initial drug-to-lipid weight ratio of 0.1:1.

FIG. 10B: A graph showing loading of daunorubicin into DSPC/DSPE-PEG2000(95:5 mole ratio) liposomes as a function of time using 150 mM CoCl2, asthe internal medium. Loading was carried out at 23° C. (•), 37° C. (∘)and 60° C. (▾) at a drug-to-lipid weight ratio of 0.1:1.

FIG. 10C: A graph showing loading of daunorubicin into DSPC/DSPE-PEG2000(95:5 mole ratio) liposomes as a function of time using 300 mM NiSO₄ asthe internal medium. Loading was carried out at 60° C. at adrug-to-lipid weight ratio of 0.2:1.

FIG. 11: A graph showing loading of epirubicin into DSPC/DSPE-PEG2000(95:5 mole ratio) liposomes as a function of time using 300 mM CuSO₄ at60° C. Epirubicin was loaded to achieve a drug-to-lipid weight ratio of0.2:1.

FIG. 12A: A graph showing loading of doxorubicin into DSPC/Chol (55:45mole ratio) liposomes as a function of time using 300 mM CoCl2 as theinternal medium and SHE, pH 7.5 as the external buffer. Loading wascarried out at 60° C. at a drug-to-lipid weight ratio of 0.1:1.

FIG. 12B: A graph showing loading of daunorubicin into DSPC/Chol (55:45mole ratio) liposomes as a function of time using 300 mM CuSO₄ as theinternal medium and HBS, pH 7.4 as the external buffer. Daunorubicin wasloaded at 60° C. at a drug-to-lipid weight ratio of 0.1:1 (•), 0.2:1 (∘)and 0.4:1 (▾).

FIG. 12C: A graph showing loading of topotecan into DSPC/DSPE-PEG (95:5mole ratio) liposomes as a function of time at 37° C. A 300 mM CuSO₄solution was used as the internal loading medium. Topotecan was loadedat a drug-to-lipid weight ratio of 0.1:1.

FIG. 13A: A graph showing loading of daunorubicin intocisplatin-containing DSPC/DSPE-PEG2000 (95:5 mole ratio) liposomes as afunction of time using 150 mM MnCl₂ as the internal medium and HBS, pH7.4 as the external solution. Cisplatin was passively encapsulated anddaunorubicin loading was carried out at 60° C. at a drug-to-lipid weightratio of 0.1:1.

FIG. 13B: A graph showing loading of daunorubicin intocisplatin-containing DMPC/Chol (55:45 mole ratio) liposomes as afunction of time using 150 mM CuCl₂ as the internal medium and HBS, pH7.4 as the external solution. Cisplatin was passively encapsulated anddaunorubicin loading was carried out at 60° C. at a drug-to-lipid weightratio of 0.1:1.

FIG. 13C: A graph showing loading of daunorubicin intocarboplatin-containing DPPC/Chol (55:45 mole ratio) liposomes as afunction of time using 300 mM NiSO₄ as the internal medium and 300 mMsucrose, 20 mM HEPES, pH 7.4 as the external solution. Carboplatin waspassively encapsulated and daunorubicin loading was carried out at 37°C. at a drug-to-lipid weight ratio of 0.1:1.

FIG. 13D: A graph showing loading of irinotecan intocisplatin-containing DPPC/Chol (55:45 mole ratio) liposomes as afunction of time using 75 mM CuCl₂+150 mM CuSO₄ as the internal mediumand SHE, pH 7.4 as the external solution. Cisplatin was passivelyencapsulated and irinotecan loading was carried out at 60° C. at adrug-to-lipid weight ratio of 0.1:1.

FIG. 14: A graph showing vincristine/lipid and doxorubicin/lipid ratiosat various time points during loading of vincristine at 50° C. intoDSPC/Chol (55:45 mole ratio) liposomes preloaded with doxorubicin.Liposomes containing 300 mM MnSO₄ were preloaded with doxorubicin (•) at50° C. at a drug-to-lipid ratio of 0.2:1 wt/wt. Vincristine loading (▪)was carried out with the aid of the A23187 ionophore at a drug-to-lipidratio of 0.05:1 wt/wt. Error bars represent the standard deviationbetween three replicate experiments.

FIG. 15: A histogram showing sequential metal loading of irinotecan anddoxorubicin into DSPC/Chol (55:45 mole ratio) liposomes containing 300mM CuSO₄ as the internal medium. Liposomes were preloaded withirinotecan at 60° C. at a drug-to-lipid mole ratio of 0.2:1 toapproximately 100% followed by encapsulation of doxorubicin, loaded at a0.15:1 drug/lipid mole ratio. As a control, liposomal uptake of eachdrug into singly loaded liposomes was measured separately. Error barsrepresent the standard deviation between three replicate experiments.

FIG. 16: A histogram showing the plasma drug-to-lipid ratio ofdaunorubicin-containing DSPC/DSPE-PEG2000 (95:5 mole ratio) liposomes 24hours after intravenous administration to Balb/c mice. Daunorubicin wasloaded at a drug-to-lipid weight ratio of 0.1:1 at 60° C. into liposomescomprising either 300 mM CuSO₄; 150 mM citrate, pH 4; or 300 mM MnSO₄ asthe internal medium. Error bars represent the standard deviation betweenthree replicate experiments.

FIG. 17: a graph showing loading of irinotecan into DSPC/DSPG (80:20 molratio) liposomes in response to encapsulated CuSO₄ following passage ofthe liposomes through a Chelex-100™ column equilibrated with 150 mMNaCl. The liposomes were subsequently exchanged into 300 mM sucrose, 20mM HEPES, pH 7.4. Loading was carried out by incubation at 37° C. (•),50° C. (∘) and 60° C. (▾).

FIG. 18: a graph showing loading of irinotecan into DSPC/DSPG (80:20 molratio) liposomes in response to encapsulated CuSO₄ a 37° C. (•), 50° C.(∘) and 60° C. (▾). The external solution of the liposome was bufferexchanged into saline and further exchanged into 300 mM sucrose, 20 mMHEPES, pH 7.4 (no external EDTA) before loading.

FIG. 19: a graph showing loading of irinotecan into DSPC/DSPG (80:20 molratio) liposomes in response to encapsulated copper gluconate afterbuffer exchange of the external solution into 300 mM sucrose, 20 mMHEPES, 30 mM EDTA, pH 7.4. Loading was carried out by incubation at 37°C. (•), 50° C. (∘) and 60° C. (▾).

FIG. 20: a graph showing loading of irinotecan into DSPC/DSPG (80:20 molratio) liposomes in response to encapsulated copper gluconate afterpassage of the liposome preparations through a Chelex-100™ columnequilibrated with 300 mM sucrose, 20 mM HEPES, pH 7.4. Loading ofirinotecan was carried out by incubation at 37° C. (•), 50° C. (∘) and60° C. (▾).

FIG. 21: a graph showing plasma lipid levels of DSPC/DSPG (80:20 molratio), DSPC/SM/DSPG (75:5:20 mol ratio) and DSPC/SM/DSPG (70:10:20 molratio) liposomes co-loaded with daunorubicin and carboplatin representedby •, ∘, and ▾ respectively. Carboplatin was passively entrapped anddaunorubicin was actively loaded in response to encapsulated CuSO₄.

FIG. 22: a graph showing plasma daunorubicin levels of DSPC/DSPG (80:20mol ratio), DSPC/SM/DSPG (75:5:20 mol ratio) and DSPC/SM/DSPG (70:10:20mol ratio) liposomes co-loaded with daunorubicin and carboplatinrepresented by •, ∘, and ▾ respectively. Carboplatin was passivelyentrapped and daunorubicin was actively loaded in response toencapsulated CuSO₄.

FIG. 23: a graph showing plasma carboplatin levels of DSPC/DSPG (80:20mol ratio), DSPC/SM/DSPG (75:5:20 mol ratio) and DSPC/SM/DSPG (70:10:20mol ratio) liposomes co-loaded with daunorubicin and carboplatinrepresented by •, ∘, and ▾ respectively. Carboplatin was passivelyentrapped and daunorubicin was actively loaded in response toencapsulated CuSO₄.

DETAILED DESCRIPTION OF THE INVENTION

Preparation of Liposomes

The term “liposome” as used herein means vesicles comprised of one ormore concentrically ordered lipid bilayers encapsulating an aqueousphase. Formation of such vesicles requires the presence of“vesicle-forming lipids” which are amphipathic lipids capable of eitherforming or being incorporated into a bilayer structure. The latter termincludes lipids that are capable of forming a bilayer by themselves orwhen in combination with another lipid or lipids. An amphipathic lipidis incorporated into a lipid bilayer by having its hydrophobic moiety incontact with the interior, hydrophobic region of the membrane bilayerand its polar head moiety oriented toward an outer, polar surface of themembrane. Hydrophilicity arises from the presence of functional groupssuch as hydroxyl, phosphato, carboxyl, sulfato, amino or sulfhydrylgroups. Hydrophobicity results from the presence of a long chain ofaliphatic hydrocarbon groups.

It will be appreciated that any suitable vesicle-forming lipid may beutilized in the practice of this invention as judged by one of skill inthe art. This includes phospholipids such as phosphatidylcholine (PC),phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid(PA), phosphatidyethanolamine (PE) and phosphatidylserine (PS);glycolipids; and sphingolipids such as sphingosine, ceramides,sphingomyelin, and glycosphingolipids (such as cerebrosides andgangliosides). Preferred phospholipids comprise two acyl chains from 6to 24 carbon atoms selected independently of one another and withvarying degrees of unsaturation.

Liposomes prepared in accordance with this invention can be generated byconventional techniques used to prepare vesicles. These techniquesinclude the ether injection method (Deamer et al., Acad. Sci. (1978)308: 250), the surfactant method (Brunner et al., Biochim. Biophys. Acta(1976) 455: 322), the freeze-thaw method (Pick et al., Arch. Biochim.Biophys. (1981) 212: 186) the reverse-phase evaporation method (Szoka etal., Biochim. Biophys. Acta. (1980) 601: 559-71), the ultrasonictreatment method (Huang et al., Biochemistry (1969) 8: 344), the ethanolinjection method (Kremer et al., Biochemistry (1977) 16: 3932), theextrusion method (Hope et al., Biochim. Biophys. Acta (1985) 812:55-65)and the french press method (Barenholz et al., FEBS Lett. (1979) 99:210). All of the above processes are basic technologies for theformation of liposome vesicles and these processes can be used incombinations. Preferably, small unilamellar vesicles (SUVs) are preparedby the ultrasonic treatment method, the ethanol injection method and theFrench press method. Preferably, multilamellar vesicles (MLVs) areprepared by the reverse-phase evaporation method or by the simpleaddition of an aqueous solution to a lipid film followed by dispersal bymechanical agitation (Bangham et al., J. Mol. Biol. (1965) 13: 238-252).

Particularly suitable liposome preparations which may be used in thepractice of this invention are large unilamellar vesicles (LUVs). LUVsmay be prepared by the ether injection method, the surfactant method,the freeze-thaw method, the reverse-phase evaporation method, the frenchpress method or the extrusion method. Preferably, LUVs are preparedaccording to the extrusion method. The extrusion method involves firstcombining lipids in chloroform to give a desired mole ratio. A lipidmarker may optionally be added to the lipid preparation. The resultingmixture is dried under a stream of nitrogen gas and placed in a vacuumpump until the solvent is substantially removed. The samples are thenhydrated in an appropriate aqueous solution, which may contain a mixtureof therapeutic agent or agents. The mixture is then passed through anextrusion apparatus (e.g. apparatus by Northern Lipids, Vancouver,Canada) to obtain liposomes of a defined size. Average liposome size canbe determined by a variety of methods including quasi-elastic lightscattering using, for example, a NICOMP™ 370 submicron particle sizer ata wavelength of 632.8 nm.

In some aspects of this invention, liposomes are prepared to be of“low-cholesterol”. Such liposomes contain “substantially nocholesterol,” or “essentially no cholesterol.” The term “substantiallyno cholesterol” allows for the presence of an amount of cholesterol thatis insufficient to significantly alter the phase transitioncharacteristics of the liposome (typically less than 20 mol %cholesterol). 20 mol % or more of cholesterol broadens the range oftemperatures at which phase transition occurs, with phase transitiondisappearing at higher cholesterol levels (e.g. greater than 30 mol %).Preferably, a liposome having substantially no cholesterol will haveabout 15 or less and more preferably about 10 or less mol % cholesterol.The term “essentially no cholesterol” means about 5 or less mol %,preferably about 2 or less mol % and even more preferably about 1 orless mol % cholesterol. Most preferably, no cholesterol will be presentor added when preparing “low cholesterol” liposomes.

Liposomes of this invention may comprise a hydrophilic polymer-lipidconjugate such as a polyalkylether-lipid conjugate. Grafting ahydrophilic polymer such as a polyalkylether to the surface of liposomeshas been utilized to “sterically stabilize” liposomes thereby increasingthe circulation longevity of liposomes. This results in enhanced bloodstability and increased circulation time, reduced uptake into healthytissues, and increased delivery to disease sites such as solid tumors(see: U.S. Pat. Nos. 5,013,556 and 5,593,622; and Patel et al., Crit RevTher Drug Carrier Syst (1992) 9: 39-90). Typically, the polymer isconjugated to a lipid component of the liposome. The term “hydrophilicpolymer-lipid conjugate” refers to a vesicle-forming lipid covalentlyjoined at its polar head moiety to a hydrophilic polymer, and istypically made from a lipid that has a reactive functional group at thepolar head moiety in order to attach the polymer. Suitable reactivefunctional groups are for example, amino, hydroxyl, carboxyl or formylgroups. The lipid may be any lipid described in the art for use in suchconjugates. Preferably, the lipid is a phospholipid having two acylchains comprising between about 6 to about 24 carbon atoms in lengthwith varying degrees of unsaturation. Most preferably, the lipid in theconjugate is a PE, preferably of the distearoyl form. The polymer is abiocompatible polymer characterized by a solubility in water thatpermits polymer chains to effectively extend away from a liposomesurface with sufficient flexibility that produces uniform surfacecoverage of a liposome. Preferably, such a polymer is a polyalkylether,including polyethylene glycol (PEG), polymethylene glycol, polyhydroxypropylene glycol, polypropylene glycol, polylactic acid, polyglycolicacid, polyacrylic acid and copolymers thereof, as well as thosedisclosed in U.S. Pat. Nos. 5,013,556 and 5,395,619. Preferably, such apolymer has a molecular weight between about 350 and 5000 daltons. Theconjugate may be prepared to include a releasable lipid-polymer linkagesuch as a peptide, ester, or disulfide linkage. The conjugate may alsoinclude a targeting ligand. Mixtures of conjugates may be incorporatedinto liposomes for use in this invention.

Negatively charged lipids as described below may be incorporated inmetal encapsulated liposome formulations to increase the circulationlongevity of the carrier. These lipids may be employed in place ofhydrophilic polymer lipid conjugates as surface stabilizing agents.Embodiments of this invention may make use of cholesterol-free liposomescontaining such negatively charged lipids to prevent aggregation therebyincreasing the blood residence time of the carrier. Such embodiments areideally loaded following rigorous removal of metal ions from the surfaceof the liposome and the external solution of the liposomes.

The term “negatively charged lipid” refers to a vesicle-forming lipidhaving one or more negative charges at physiological pH, includingphospholipids and sphingolipids. Negatively charged lipids may beincorporated in a liposome of this invention at 5 to 95 mol %, morepreferably at 10 to 50 mol % and most preferably at 15 to 30 mol %.

Preferably, a lipid that is negatively charged at physiological pH foruse in this invention will comprise a “non-zwitterionic moiety” whichrefers to a moiety that does not have opposing charges at physiologicalpH. Such lipids impart to the liposome desirable circulation propertiesfor in vivo uses. The net negative charge on the lipid may arise solelyfrom the presence of the negative charge on the lipid (e.g. from aphosphate group) or where the lipid has more than one charge, additionalnegative charge may be due to the presence of a negatively chargednon-zwitterionic moiety. Preferably, however, the negative charge arisessolely from the lipid component in which case the non-zwitterionicmoiety is a neutral group. Preferably, the non-zwitterionic comprises 2to 6 carbon atoms.

Suitable non-zwitterionic moieties contain electron-withdrawingfunctional groups that impart to the head group hydrophiliccharacteristics. Such functional groups can be selected from the groupconsisting of alcohols, acids, ketones, esters, ethers, amides andaldehydes. Non-zwitterionic moieties of the following formulas may beutilized:

Alcohols

P—R or POR or PO(CH₂)₂NHR where R is—(CH₂)_(v)(CH)_(w)(C)_(x)(OH)_(y)(CH₃)_(z)

wherein the number of carbons (v+w+x+z) is 2-6 most preferably 3-5

where the number of OH groups is 1-3 (y=1-3)

e.g. DPPG

Ketones

P—R or POR or PO(CH₂)₂NHR where R is —(CH₂)_(v)(C)_(x)(CO)_(y)(CH₃)_(z)

where the number of carbons (v+x+y+z) is 2-6 most preferably 3-5

where the number of ketone groups is 1-2 (y=1-2)

e.g. N-butyryl-DPPE, N-valeryl-DPPE

Carboxylic Acids

P—R or POR or PO(CH₂)₂NHR where R is—(CH₂)_(u)(CH)_(v)(C)_(x)(COOH)_(y)(CH₃)_(z)

where the number of carbons (u+v+x+y+z) is 2-6 most preferably 3-5

where the number of carboxylic acid groups is 1-2 (y=1-2)

Esters

P—R or POR or PO(CH₂)₂NHR where R is —(CH₂)_(v)(C)_(x)(COO)_(y)(CH₃)_(z)

where the number of carbons (v+x+y+z) is 2-6 most preferably 3-5

where the number of ester groups is 1-2 (y=1-2)

Ethers

P—R or POR or PO(CH₂)₂NHR where R is —(CH₂)_(v)(C)_(w)(O)_(y)(CH₃)_(z)

where the number of carbons (v+x+z) is 2-6 most preferably 3-5

where the number of ether groups is 1-2 (y=1-2)

Amines

Primary Amines:

P—R or POR or PO(CH²)₂NHR where R is—(CH₂)_(v)(C)_(w)(CH)_(x)(NH₃)_(y)(CH₃)_(z)

where the number of carbons (v+w+x+z) is 2-6 most preferably 3-5

where the number of amino groups is 1-2 (y=1-2)

Secondary Amines:

P—R or POR or PO(CH₂)₂NHR where R is—(CH₂)_(v)(C)_(w)(CH)_(x)(NH₂)_(y)(CH₃)_(z)

where the number of carbons (v+w+x+z) is 2-6 most preferably 3-5

where the number of amine groups is 1-2 (y=1-2)

Tertiary Amines:

P—R or POR or PO(CH₂)₂NHR where R is—(CH₂)_(v)(CH)_(w)(C)_(x)(N)_(y)(CH₃)_(z)

where the number of carbons (v+w+x+z) is 2-6 most preferably 3-5

-   -   where the number of amine groups is 1(y=1)

The non-zwitterionic moiety may also be comprised of combinations offunctional groups; for example a compound of formula:

Carboxylic Acids and Ketones

P—R or POR or PO(CH₂)₂NHR where R is—(CH₂)_(u)(CH)_(v)(C)_(w)(COOH)_(x)(CO)_(y)(CH₃)_(z)

where the number of carbons (u+v+w+x+y+z) is 2-6 most preferably 3-5

where the number of carboxylic acid groups is 1-2 (x=1-2)

where the number of ketone groups is 1-2 (y=1-2)

e.g. N-succinyl-DPPE, N-glutaryl-DPPE

P—R or POR or PO(CH₂)₂NHR where R is—(CH₂)_(s)(CH)_(t)(C)_(u)(COOH)_(v)(CO)_(x)(OH)_(y)(CH₃)_(z)

Carboxylic Acids, Ketones and Alcohols

where the number of carbons (s+t+u+v+x+z) is 2-6 most preferably 3-5

where the number of carboxylic acid groups is 1-2 (v=1-2)

where the number of ketone groups is 1-2 (x=1-2)

where the number of hydroxyl groups is 1-2 (y=1-2)

e.g. N-tartaryl-DPPE

Ring Structures

P—R or POR or PO(CH₂)₂NHR where R is a 5 or 6 member ring containing 1-5or 1-6 alcohol groups (cyclitols), respectively (e.g.phosphatidylinositol).

Carbohydrates

Monosaccharides that may be used in the practice of this inventioninclude arabinose, fucose, galactose, glucose, lyxose, ribose andxylose. Disaccharides include sucrose, lactose, trehalose, cellobiose,gentiobiose and maltose. For purposes of extending the circulationlifetime of the liposome, monosaccharides and disaccharides which do notbind to cellular receptors are preferred (e.g. mannose).

In the case where the non-zwitterionic moiety is neutral, the head groupconsists of groups that are neutral at physiological pH includingalcohols, ketones, esters, ethers, amides and aldehydes.

In preferred embodiments of the invention, the non-zwitterionic moietyis a short-chain alcohol, a preferred alcohol containing two or morehydroxyl groups. The alcohol can be a straight-chain polyol of whichglycerol is an example. Glycerol may make up the head group of aphosphosphingolipid or a phospholipid through linkage of one of thehydroxyl groups to the phosphate group of the lipid. Most preferably,glycerol is attached to the phosphate via a terminal hydroxyl group ofthe glycerol molecule, the resulting molecule being termedphosphatidylglycerol (PG). Preferably the fatty acid chains of thephosphatidylglycerol are selected independently of each other from thegroup consisting of caproyl (6:0), octanoyl (8:0), capryl (10:0),lauroyl (12:0), myristoyl (14:0), palmitoyl (16:0), stearoyl (18:0),arachidoyl (20:0), behenoyl (22:0), lingnoceroyl (24:0) and phytanoyl,including the unsaturated versions of these fatty acid chains in the cisor trans configurations such as oleoyl (18:1), linoleoyl (18:2),arachidonoyl (20:4) and docosahexaenoyl (22:6). Phospholipids having twoacyl chains of 14 to 18 carbon atoms are preferred.

In another preferred embodiment of the invention, the non-zwitterionicmoiety is a ring structure. Most preferably the ring structure is acyclitol, which is a cycloalkane containing one hydroxyl group on eachof three or more ring atoms. Such compounds may be derivatived withvarious groups to impart to the molecule a desired water solubility.Preferably the cyclitol is an inositol attached to a phospholipidthrough the phosphate group, the resulting compound beingphosphatidylinositol (PI). Preferably, the fatty acid chains of thephosphatidylinositol are selected independently of each other from thegroup consisting of caproyl (6:0), octanoyl (8:0), capryl (10:0),lauroyl (12:0), myristoyl (14:0), palmitoyl (16:0), stearoyl (18:0),arachidoyl (20:0), behenoyl (22:0), lingnoceroyl (24:0) and phytanoyl,including the unsaturated versions of these fatty acid chains in the cisor trans configurations such as oleoyl (18:1), linoleoyl (18:2),arachidonoyl (20:4) and docosahexaenoyl (22:6). Phosphatidylinositolhaving two acyl chains of 14 to 18 carbon atoms are preferred.

Negatively charged lipids may be obtained from natural sources or may bechemically synthesized. Methods to covalently attach compounds to thehead group of a lipid are well known in the art and generally involvereacting functional groups on the terminal portion of the lipid headgroup with functional groups on the moiety to be attached. Suitablelipids for the chemical attachment of a hydrophilic moiety includelipids having a polar head group that terminates with a reactivefunctional group such as an amine or a carboxylic acid. An example of aparticularly suitable lipid is phosphatidylethanolamine as it contains areactive amino group. Methods for preparing phosphatidylethanolaminederivatives have been described in Ahl, P., et al. (1997) Biochimica etBiophysica Act 1329: 370-382, the reference of which is incorporatedherein by reference. Examples of negatively charged lipids obtained fromnatural sources include phosphatidylglycerol and phosphatidylinositolobtained from egg and plant sources respectively.

Encapsulation of Active Agents and Metals in Liposomes

This invention provides a method for loading an agent into a liposomecomprising an encapsulated transition metal. Within this specification,the term “agent” refers to substances which are capable of beingencapsulated into liposomes according to this invention. Preferably,such an agent will be a “therapeutic agent” capable of exerting aneffect on a target, in vitro or in vivo. Suitable active agents include,for example, prodrugs, diagnostic agents, therapeutic agents,pharmaceutical agents, drugs, synthetic organic molecules, proteins,peptides, vitamins, steroids and steroid analogs. The agent, at leastwhen not complexed with a transition metal, must be permeable across aliposomal membrane in order to achieve loading.

Transition metals for use in this invention include the Group 1B, 2B,3B, 4B, 5B, 6B, 7B and 8B elements (groups 3-12). Preferred metalsinclude those selected from the group consisting of Fe, Co, Ni, Cu, Zn,V, Ti, Cr, Rh, Ru, Mo, Mn and Pd. More preferably, the metal is Fe, Co,Ni, Cu, Mn or Zn. Even more preferably, the metal is Zn, Mn, Co or Cu.Even more preferably, the metal is Zn, Co, or Cu.

Transition metal ions used in accordance with this invention may beencapsulated in liposomes according to conventional techniques known inthe art. This includes the passive encapsulation techniques known in theart and as described below.

Preferably, the liposomes are formed in a solution comprising atransition metal at a concentration of from about 20 mM to about 1 M,preferably from about 50 mM to about 800 mM and more preferably fromabout 100 to about 350 mM.

Various salts of metals may be employed in the practice of thisinvention. Preferably, the salt is pharmaceutically acceptable andsoluble in aqueous solvent. Preferred salts may be selected from thegroup consisting of chlorides, sulfates, tartrates, citrates,phosphates, nitrates, carbonates, acetates, glutamates, gluconates,glycinates, histidinates, lysinates and the like.

Preferably, a therapeutic agent to be encapsulated into a liposome ofthis invention is one which is capable of coordinating with a metalencapsulated in the liposome. Agents that are capable of coordinatingwith a transition metal typically comprise coordination sites such asamines, carbonyl groups, ethers, ketones, acyl groups, acetylenes,olefins, thiols, hydroxyl, halides, groups or other suitable groupscapable of donating electrons to the transition metal thereby forming acomplex with the metal. Examples of agents which bind transition metalsand thus may be used in the practice of this invention includequinolones such as fluoroquinolones, quionlones such as nalidixic acid,anthracyclines such as doxorubicin, daunorubicin idarubicin andepirubicin, amino glycosides such as kanamycin and other antibioticssuch as bleomycin, mitomycin C and tetracycline and nitrogen mustardssuch as cyclophosphamide, thiosemicarbazones, indomethacin andnitroprusside, camptothecins such as topotecan, irinotecan, lurtotecan,9-aminocamptothecin, 9-nitrocamptothecin and 10-hydroxycamptothecin andpodophyllotoxins such as etoposide. Agents used in this invention can becapable of donating electrons from different atoms in the agent and todifferent sites in the geometric structure of the complex. Such agentscapable of donating more than one non-bonding pair of electrons are alsoknown as multidentate. Preferably a therapeutic agent for use in thisinvention is an antineoplastic agent.

Non-limiting examples of active agents that complex with transitionmetals and thus may be used in the practice of this invention areprovided in Table I. TABLE I EXAMPLES OF METAL-BASED ACTIVE AGENTSMETAL(S) AGENT(S) REFERENCE Cu etoposide Tawa et al. (1997) Biol. Pharm.Bull. 20: 1002-1005 Fe(III) dexrazoxane, Hasinoff et al. (1999) Journalof Inorganic Biochemistry 77: 257-259 losoxantrone, piroxantrone Zn,Cu(II), bleomycin Wenbao et al. (2001) Biochemistry 40: 7559-7568Fe(III), Co(III) Fe(III) anthracyclines Fiallo et al. (1999) Journal ofInorganic Biochemistry 75: 105-115 Bi(III) quinolones Turel et al.(1997) Journal of Inorganic Biochemistry 66: 241-245 Cu(II) L-lysineChikira et al. (1997) Journal of Inorganic Biochemistry 66: 131-139L-arginine Cu(II), Ni(II), desferrioxamine B Farkas et al. (1997)Journal of Inorganic Biochemistry 65: 281-286 Zn(II), MoVI) Cu(II)cynnamyl Bontchev et al. (1997) Journal of Inorganic Biochemistry 65:175-182 derivative of rafamycin Fe(III) adriamycin Capolongo et al.(1997) Journal of Inorganic Biochemistry 65: 115-122 Cu(II), Ni(II)cinoxacin Ruiz et al. (1997) Journal of Inorganic Biochemistry 65: 87-96

Methods of determining whether coordination occurs between an agent anda transition metal include conventional techniques well know to those ofskill in the art. Preferred techniques involve measuring the absorptionspectra or using NMR as described by Greenaway and Dabrowiak (J. Inorg.Biochem. (1982) 16(2): 91). If desired, an active agent may be testedbefore encapsulation in order to determine whether coordination occursand the optimal pH for complexation.

A preferred technique for preparing liposomes with an encapsulated metalinvolves first combining lipids in chloroform to give a desired moleratio. A lipid marker may optionally be added to the lipid preparation.The resulting mixture is dried under a stream of nitrogen gas and placedin a vacuum pump until the solvent is removed. Subsequently, the samplesare hydrated in a solution comprising a transition metal (which maycomprise more than one metal, for example Cu and Mn, or one metal, butdifferent salts of the metal). The mixture is then passed through anextrusion apparatus to obtain a preparation of liposomes of a definedsize. Average liposome size can be determined by quasi-elastic lightscattering using a NICOMP™ 370 submicron particle sizer at a wavelengthof 632.8 nm. Subsequent to extrusion, the external solution may betreated or replaced so as to remove metal ions from the externalsolution and the liposome surface.

This invention preferably makes use of liposomes with an encapsulated or“internal” medium comprising a transition metal in a “metal compatiblesolution”. Use of a metal compatible solution prevents precipitation ofthe metal or minimizes precipitation to an extent sufficient to allowfor pharmaceutical use of the liposomes.

A metal compatible solution is defined as one that consists of a metalin solution that does not cause unacceptable precipitation to occur forat least the time required to formulate liposomes. Preferably, the metalsolution should be clear and soluble, free of aggregation, precipitationor flocculation for at least about 4 hours. By way of example, a 300 mMsolution of MnSO₄ in pH 7.4 HEPES buffer as described in Cheung, et al.[supra] is not a metal compatible solution as it produces an obviousbrown precipitate of Mn(OH)2 comprising approximately 6-7 molar % of themanganese added to the solution.

Various methods are known in the art and may be used to determine if themetal solution is forming a precipitate such as centrifugation of thesolution and an evaluation of whether a pellet is formed or observationof cloudiness in the solution. The absorbance of the solution can alsobe monitored by spectroscopy (e.g. increase in absorbance at 690 nm),where a substantial increase in absorbance is indicative of solutioninstability and precipitation. The simplest method is to filter thesolution and look for the presence of a precipitate on the filter. Forexample, a 50 ml sample may be passed through Whatman(™) No. 2 filterpaper and the filter observed for visible sediment.

A preferred method to determine whether a solution is metal compatibleis to monitor absorbance at 690 nm. Additional of metal should notresult in an increase of more than about 0.1 absorption units andpreferably no more than about 0.05 units.

An alternative preferred method of determining whether a metal solutionis metal compatible is by centrifugation (e.g. 100 ml sample at 1000 rpmfor 10 minutes) to collect any precipitate, measuring the amount ofprecipitate collected and determining the proportion of the metal addedto the original solution present in the precipitate. The amount of metalin the precipitate should not exceed about 1 molar % of the amount ofmetal added to the original solution.

Preferred metal compatible solutions are those that are alsopharmaceutically acceptable such as ones comprising triethanolamine(TEA), sodium chloride, sodium acetate/acetic acid, sodiumcitrate/citric acid or sugars such as sucrose, dextrose and lactose.Phosphate and carbonate based solutions (although pharmaceuticallyacceptable) will have limited use except at pH's outside of normalphysiological ranges, due to the likelihood of metal precipitation.Preferably, the metal compatible solution is buffered and has pH in aphysiological range.

In the practice of this invention, it may be advantageous for theexternal solution of the liposome preparation to be replaced or betreated in order that the resulting external solution containsubstantially no uncomplexed metal ions prior to loading of an agent.For purposes of this specification, “uncomplexed metal ions” includesmetal ions free in the external solution and metal ions bound to (orotherwise associated with) the external surface of the liposomes.Conversely, a complexed metal ion is one which is no longer free tointeract with the therapeutic agent or the liposome surface because itis present in the external solution in a complex with a moiety such as achelating agent. Thus, it is preferable that the surface of theliposomes and the external solution be substantially free of the metalions or if metal ions are present, that they be complexed with achelating agent. Examples of cationic chelating agents that may beemployed include: EDTA and derivatives; EGTA and derivatives; histidine;Chelex(™); TPEN and derivatives; BAPTA and derivatives; bishosphonate;o-phenanthrolene (phenanthroline); citrate; InsP6; Diazo-2; and DTPA(diethylene-triaminopenta acetic acid) isothiocyanate.

Replacement of the external solution to remove metal ions can beaccomplished by various other techniques, such as by chromatography ofthe liposome preparation through an extensive gel filtration columnequilibrated with a second aqueous buffered solution, by centrifugation,extensive or repeated dialysis, exchange of the external medium,treating the external solution with chelating agents or by relatedtechniques. A single solution exchange or round of dialysis without theuse of a chelating agent is typically insufficient to remove metal ionsfrom the surface of negatively charged liposomes.

The external solution is also preferably a buffered solution. However,it is appreciated that any suitable solvent may be utilized in thepractice of this invention. A preferred external solution has a pH atabout physiological pH and comprises a buffer which has a bufferingrange to include physiological pH. Non-limiting examples of suitablebuffers for the external solution are HBS, pH 7.4 (150 mM NaCl, 20 mMHEPES) and SHE, pH 7.4 (300 mM sucrose, 20 mM HEPES, 30 mM EDTA).

Uptake of an agent may be established by incubation of the mixture at asuitable temperature after addition of the agent to the external medium.Depending on the composition of the liposome, temperature and pH of theinternal medium, and chemical nature of the agent, uptake of the agentmay occur over a time period of minutes or hours. Loading may be carriedout at temperatures of, for example, 20° C. to about 75° C., preferablyfrom about 30° C. to about 60° C.

Removal of unencapsulated agent may be carried out by passing a liposomepreparation through a gel filtration column equilibrated with a secondaqueous buffered solution, or by centrifugation, dialysis, or relatedtechniques. Preferably, the second solution is one that isphysiologically compatible but need not be “metal compatible”. Afterremoval of unencapsulated active agent, the extent of agent loading maybe determined by measurement of drug and lipid levels according toconventional techniques. Lipid and drug concentrations may be determinedby employing techniques such as scintillation counting,spectrophotometric assays, fluorescent assays and high performanceliquid chromatography. The choice of analysis depends on the nature ofthe drug and whether the liposomes contain a radiolabeled lipid marker.An example of quantification utilizing a radiolabeled marker is setforth in the Examples herein, although it will be appreciated that anysuitable method of determining the extent of loading may be used.

Prior to loading of an agent into a liposome using an encapsulatedtransition metal, the liposome may be passively co-encapsulated with anagent and a metal. Using this approach, two or more agents may beincorporated into the liposome by combining passive and active methodsof loading.

Subsequent to loading of an agent into a liposome, an ionophore may beincubated with the mixture such that insertion of the ionophore into thebilayer occurs. The term “ionophore” refers to a compound which forms acomplex with a metal ion and assists the ion in crossing a lipid bilayerwhile further assisting the transport of H+ in the counter direction.Examples of suitable ionophores for the present invention includenigericin, monensin, dianemycin, A23187, 4-BrA23187, ionomycin andX-537A. The ionophores may be specific for monovalent or divalent metalions. Examples of ionophores specific for monovalent metal ions includenigericin, monensin and dianemycin. Uptake of the ionophore isestablished by addition of the ionophore to the mixture and incubationat a temperature suitable for incorporation of the ionophore into theliposomal bilayer. The amount of ionophore used will typically depend onthe nature and type of liposome formulation. Addition of the ionophoreto the liposome after loading of the agent may be carried out in orderto subsequently impose a pH gradient across the liposomal bilayer toalter the retention properties of the agent in the liposome or toprotect agents that are affected by neutral or alkaline environmentssuch as, topotecan and irinotecan.

Preferred metal compatible solutions may include components such asbuffers that can be utilized between pH 6.0 and 8.5. Preferably, thebuffer does not substantially precipitate over a two-day time period at4° C. with an encapsulated metal ion at pH 6.0 to 8.0 and morepreferably pH 6.5 to 7.5. A buffer may be tested for its ability toprevent precipitation by visually inspecting the solution for theappearance of cloudiness, which is indicative of formation of aprecipitate. An example of a method for determining whether a buffer iscompatible with a particular transition metal is outlined in Example 3.After encapsulation of a transition metal in a metal compatiblesolution, an agent may be added to the external medium such that theagent is encapsulated into the liposome. Liposomes encapsulating atransition metal and a metal compatible solution may be preparedaccording to conventional techniques known in the art including thetechniques described above. It is appreciated, however, that anysuitable metal may be utilized in this aspect of the invention.Preferably, the liposome with the encapsulated agent or agents has anextraliposomal pH that is substantially similar to the intraliposomalpH. Most preferably, the extraliposomal and intraliposomal pH is aboutpH 6.0 to pH 8.0, most preferably, it is between about pH 6.5 and pH7.5.

The present invention further provides a method of designing liposomes,said method comprising selecting a metal ion for encapsulation in aliposome to achieve a desired retention of an encapsulated agent. Itwill be appreciated that any suitable liposome and agent may be utilizedin the practice of this aspect of the invention. Other preferredfeatures and conditions of this aspect of the invention are as generallydescribed above.

In order to determine the rate of release of an agent from a liposome,the liposome may be administered intravenously and plasma levels ofagent and lipid measured after administration. For example, the lipidcomponent may be radioactively labeled and the plasma subjected toliquid scintillation counting. The amount of drug may be determined by aspectrophotometric, HPLC or other assays. Similarly, testing for theretention of the agent in the liposome may be carried out in vitro inplasma or a suitable buffer. By way of example, a liposome comprising anencapsulated agent and transition metal may be tested in vitro or invivo for retention of agent. If a desired retention of the agent is notachieved, a different metal may be selected and tested for its abilityto retain the agent of interest.

Administering Liposomes

This invention also relates to methods of administering liposomes to amammal, and methods of treating a mammal affected by or susceptible toor suspected of being affected by a disorder (e.g. cancer). Methods oftreatment or of administration will generally be understood to compriseadministering the pharmaceutical composition at a dosage sufficient toameliorate said disorder or symptoms thereof.

For treatment of human ailments, a qualified physician may be expectedto determine how the compositions of the present invention should beutilized with respect to dose, schedule and route of administrationusing established protocols. Such applications may also utilize doseescalation should active agents encapsulated in delivery vehiclecompositions of the present invention exhibit reduced toxicity tohealthy tissues of the subject.

Preferably, the pharmaceutical compositions are administeredparenterally, i.e., intraarterialy, intravenously, intraperitoneally,subcutaneously, or intramuscularly or via aerosol. Aerosoladministration methods include intranasal and pulmonary administration.More preferably, the pharmaceutical compositions are administeredintravenously or intraperitoneally by a bolus injection or infusion. Forexample, see Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat.No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871;Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No.4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578. Particularformulations which are suitable for this use are found in Remington'sPharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985).

EXAMPLES

The following examples are given for the purpose of illustration and arenot by way of limitation on the scope of the invention. Unless otherwisespecified, pH was adjusted using triethanolamine (TEA) and results shownin the drawings are from a single representative example.

Methods for Preparation of Large Unilamellar Liposomes

Lipids were dissolved in chloroform solution and subsequently driedunder a stream of nitrogen gas and placed in a vacuum pump to removesolvent. Unless otherwise specified, trace levels of radioactive lipid3H-CHE were added to quantify lipid during the formulation process. Theresulting lipid film was placed under high vacuum for a minimum of 2hours. The lipid film was hydrated in the solution indicated to formmultilamellar vesicles (MLVs). The resulting preparation was extruded 10times through stacked polycarbonate filters with an extrusion apparatus(Lipex Biomembranes, Vancouver, BC) to achieve a mean liposome sizebetween 80 and 150 nm. All constituent lipids of liposomes are reportedin mole %.

Methods for Quantification of Drug Loading

At various time points after initiation of drug loading, aliquots wereremoved and passed through a Sephadex G-50 spin column to separate freefrom encapsulated drug. To a specified volume of eluant, Triton X-100 orN-ocyl beta-D-glucopyranoside (OGP) was added to solubilize theliposomes. Following addition of detergent, the mixture was heated tothe cloud point of the detergent and allowed to cool to room temperaturebefore measurement of the absorbance or fluorescence. Drugconcentrations were calculated by comparison to a standard curve. Lipidlevels were measured by liquid scintillation counting.

Example 1 Metal Loading can Occur in the Absence of a pH Gradient

Metal-containing liposomes with internal and external solutions bufferedto pH 7.4 were investigated for their ability to load drug. Thesestudies were performed to determine whether metal-based loading of drugcould occur independently of the presence of a pH gradient. Conventionaltechniques for actively loading drugs into liposomes often require thepresence of a transmembrane pH gradient.

In order to determine whether copper loading of irinotecan in theabsence of a pH gradient could occur using a cholesterol-freeformulation, DSPC/DSPG (80:20 mole ratio) liposomes containingcopper(II)gluconate were prepared with an external and internal pH of7.4. Lipid films of DSPC/DSPG at a mole ratio of 80:20 were prepared asdescribed above in the method section. The lipid films were hydrated in100 mM Cu(II)gluconate adjusted to pH 7.4 with triethanolamine (TEA) andextruded at 70° C. The liposomes were buffer exchanged into 300 mMsucrose, 20 mM HEPES, 30 mM EDTA (SHE buffer), pH 7.4 by tangential flowdialysis and subsequently washed three times in 6 mL of SHE, pH 7.4 toremove any copper(II)gluconate from the extraliposomal solution.Irinotecan was added to the liposome preparation at a 0.1:1drug-to-lipid mole ratio and incubated at 50° C. The extent of drugloading was determined as described in the methods by measuringabsorbance at 370 nm and lipid levels were determined by liquidscintillation counting.

Results depicted in FIG. 1A show that loading of irinotecan intoDSPC/DSPG (80:20 mole ratio) liposomes with no pH gradient at 50° C. wasessentially complete within about 5 minutes after initiation of loading.

Loading of daunorubicin into DSPC/DSPG (90:10 mole ratio) liposomescontaining encapsulated CuSO₄ buffered to pH 7.4 was also investigated.Lipid films were prepared according to the methods except DSPG wasdissolved in chloroform/methanol/water (50:10:1 v/v). A solution of 150mM CuSO₄, 20 mM histidine (adjusted to pH 7.4 using TEA), was employedas the hydration medium and MLVs were extruded at 70° C. The liposomeswere exchanged into SHE, pH 7.4 using a hand-held tangential flowdialysis column. Daunorubicin was loaded at a 0.1:1 drug/lipid weightratio. A drug-to-lipid ratio at various time points during loading wasdetermined by measuring absorbance at 480 nm after solubilization indetergent to quantify daunorubicin as described; lipid levels weredetermined by liquid scintillation counting.

As summarized in FIG. 1B, uptake of daunorubicin into DSPC/DSPG (90:10mole ratio) liposomes in the absence of a pH gradient was 100% at alltime points measured.

Copper loading of irinotecan into cholesterol-containing liposomesexhibiting no pH gradient was investigated employing DPPC/Chol (55:45mole ratio) liposomes. The liposomes were prepared as described in themethods by hydrating lipid films in a solution of 100 mMcopper(II)gluconate adjusted to pH 7.4 with TEA. Liposomes were extrudedat 65° C. and the external buffer of the liposomes was exchanged to SHE,pH 7.4 by tangential flow dialysis. Liposomes were incubated withirinotecan at a 0.1:1 drug-to-lipid weight ratio at 50° C. and theextent of drug loading was determined as described by measuringabsorbance at 370 nm after solubilization by detergent.

Loading of irinotecan into DPPC/Chol (55:45 mole ratio) liposomes in theabsence of a pH gradient revealed that almost complete loading wasobserved after about 60 minutes of incubation (FIG. 2).

In addition to copper loading, loading of drug using MnSO₄ containingliposomes in the absence of a pH gradient was also investigated.DSPC/DSPE-PEG2000 (95:5 mole ratio) liposomes were prepared with aninternal MnSO₄ solution buffered to pH 7.4 and an external solutionbuffered to pH 7.4 with SHE. Lipids films were prepared as described andhydrated in 300 mM MnSO₄ buffered to pH 7.4 with 20 mM imidazole(initial pH was adjusted to 7.4 with concentrated HC1) and extrusion wascarried out at 70° C. The samples were run down a Sephadex G-50 columnto exchange the exterior buffer with SHE, pH 7.4. Epirubicin was loadedat a drug-to-lipid weight ratio of 0.2:1 and loading was carried out at60° C. The extent of drug loading was measured as described in themethods by measuring drug absorbance at 480 nm.

Results summarized in FIG. 3 reveal that manganese loading of epirubicininto DSPC/DSPE-PEG2000 (95:5 mole ratio) liposomes at 60° C. does notrequire the presence of a pH gradient as efficient encapsulation of drugoccurred at each time point measured.

Example 2 Metal Loading of a Second Drug into Buffered LiposomesContaining a Passively Encapsulated First Drug

Although the above examples describe the metal-induced loading of onedrug into liposomes, the technique can be employed to load two or moredrugs into a single liposome. One technique involves first passivelyentrapping at least one drug along with a metal during preparation ofthe liposome followed by active metal loading of another drug. In thisexample, liposomes were prepared such that there was no pH gradientacross the membrane thus ensuring loading of the second drug by theprocess of this invention.

Loading of irinotecan into DSPC/DSPG and DSPC/Chol/DSPG liposomes,containing passively encapsulated floxuridine (FUDR), was investigatedusing various conditions as well as loading of irinotecan intocarboplatin-containing liposomes and daunorubicin loading intocisplatin-containing liposomes.

DSPC/DSPG (85:15 mole ratio) liposomes containing FUDR were prepared bydissolving DSPC in chloroform and DSPG in chloroform/methanol/water(50:10:1 v/v). The lipids were then combined together at an 85:15 moleratio and labeled with trace amounts of 14C-CHE. The samples werehydrated in 100 mM copper(II)gluconate, 220 mM TEA, pH 7.4, containing24.62 mg/mL (100 mM) FUDR with trace levels of 3H-FUDR at 70° C. Theresulting MLVs were extruded at 70° C., then buffer exchanged first intosaline and next into SHE, pH 7.4 using a hand-held tangential flowdialysis column. This sample was then exchanged into 300 mM sucrose, 20mM HEPES, pH 7.4 to remove any EDTA in the exterior buffer.

Irinotecan was added to the resulting liposome preparation at adrug-to-lipid mole ratio of 0.1:1 at 50° C. A drug-to-lipid ratio forthe spun column eluant was generated using liquid scintillation countingto determine lipid and FUDR concentrations, and absorbance at 370 nm todetermine irinotecan concentrations. Prior to measurement of absorbance,liposomes were solubilized in a solution containing Triton X-100. Theinitial FUDR drug-to-lipid mole ratio was 0.09:1, and 0.06:1 afterloading of irinotecan occurred.

FIG. 4A shows that loading of irinotecan into cholesterol-free DSPC/DSPG(85:15 mole ratio) liposomes containing encapsulated FUDR and metal doesnot require the presence of a pH gradient as efficient loading of thedrug occurred throughout the time course of the experiment.

DSPC/Chol/DSPG (70:10:20 mole ratio) liposomes containing FUDR andcopper(II)gluconate were prepared as described above. To measure theeffects of external buffer on loading, half of the resulting LUVs werebuffer exchanged into SHE, pH 7.4 and then into 20 mM HEPES, 150 mM NaCl(HBS), pH 7.4 while the other half was further exchanged into 300 mMsucrose, 20 mM HEPES, pH 7.4 using a hand-held tangential flow dialysiscolumn. Irinotecan was added to the FUDR-containing liposomes, andsubsequently measured, as described above. The initial FUDRdrug-to-lipid mole ratios were 0.1:1 and 0.09:1 for samples respectivelycontaining HBS (closed circles) or 300 mM sucrose, 20 mM HEPES (opencircles) as the external buffer. After loading of irinotecan, the samesamples had FUDR drug/lipid ratios of 0.09:1 and 0.08:1, respectively.

Results summarized in FIG. 4B show that irinotecan efficiently loadsinto low cholesterol-containing liposomes with encapsulated FUDRregardless of the external buffer employed. Loading in the absence of apH gradient further supports that this degree of irinotecan uptakeoccurs through the metal loading technique of this invention.

We have also shown various other drugs capable of metal loading in theabsence of a pH gradient into liposomes containing passivelyencapsulated drug; examples are detailed as follows:

Loading of irinotecan into DSPC/DSPG (80:20 mole ratio) liposomes withpassively encapsulated carboplatin was measured using liposomes preparedas described above except that lipid films were hydrated in 150 mM CUSO₄(adjusted to pH 7.4 using TEA), containing 25 mg/ml carboplatin. Sampleswere extruded and external buffers exchanged into SHE, pH 7.4, using ahand-held tangential flow dialysis column. Irinotecan was added at 60°C. at a drug-to-lipid weight ratio of 0.1:1 and uptake was measured aspreviously described. Atomic absorption spectrometry (AA) was used todetermine carboplatin concentrations and absorbance at 370 nm wasmeasured to determine irinotecan concentrations. The initial carboplatindrug-to-lipid weight ratio was 0.030, and 0.025 after loading ofirinotecan occurred.

As seen in the graph of FIG. 5, irinotecan loads to a high degree incarboplatin and metal-containing DSPC/DSPG (80:20 mole ratio) liposomesin the absence of a pH gradient.

To measure loading of daunorubicin into liposomes containingencapsulated cisplatin, DSPC/Chol (55:45 mole ratio) liposomes wereprepared as described for FIG. 4B except that lipid films were hydratedwith a cisplatin solution. Solid Cisplatin (40 mg/mL) was dissolved in150 mM CuCl₂, pH 7.4 (pH adjusted with NaOH) with the addition of 4%DMSO at 80° C. then added to the lipid films and allowed to hydrate at80° C. with frequent vortexing. Upon cooling, the samples werecentrifuged on a bench top centrifuge to pellet any unencapsulatedcisplatin, and the supernatant collected. The liposomes were thenapplied to a Sephadex G-50 column pre-equilibrated with HBS, pH 7.4 toremove excess metal ions from the outside of the liposomes.

Daunorubicin was added to the liposomes at a 0.1:1 weight ratio andloading was carried out at 60° C. Aliquots were removed at various timepoints and applied to a Sephadex G-50 spin column. Absorbancemeasurements were carried out at 480 nm was used to determinedaunorubicin concentrations and cisplatin levels were measured using AA.The initial cisplatin drug-to-lipid ratio was 0.044:1.

FIG. 6 shows that DSPC/Chol liposomes containing passively encapsulatedcisplatin efficiently load daunorubicin in the absence of a pH gradient.This further supports loading of a second agent, into liposomes, throughmetal loading complexation.

Example 3 The Effect of Buffer Composition on the Precipitation of Metalion

Solutions of cobalt, nickel, manganese, cadmium, zinc and copper wereprepared at concentrations of 150 and 300 mM in 20 mM histidine.Triethanolamine (1.13 g/mL) was added drop-wise until the resultingsolution was pH 7.4 or until the solution was cloudy in appearance (overa 10 minute observation period). Typically, less than 500 (L of 1.13g/mL triethanolamine was added. Subsequent to addition oftriethanolamine, the solutions were visually inspected to determinewhether precipitation of the metal had occurred. A cloudy appearance ofthe solution indicated the presence of a precipitate whereas clarity ofthe solution indicated a lack of precipitation. The results are shown inTable 2. TABLE 2 Metal Concentration (mM) Sulfate Chloride NitrateCobalt 300 no ppt no ppt ------ 150 no ppt no ppt ------ Nickel 300 noppt no ppt ------ 150 no ppt no ppt ------ Manganese 300 no ppt ppt------ 150 no ppt ppt ------ Cadmium 300 no ppt ppt ------ 150 no pptppt ------ Zinc 300 ppt ppt ------ 150 ppt ppt ------ Copper 300 no pptno ppt no ppt 150 no ppt no ppt ------ppt: represents that the formation of a precipitate occurred after theaddition of triethanolamine within a time course of 10 minutesno ppt: represents that the formation of a precipitate did not occurafter addition of triethanolamine to achieve a pH of 7.4 and within atime course of 10 minutes.dashed line: not measuredConcentrations of the indicated metal are concentrations before additionof triethanolamine.

Example 4 Metal Loading is Distinct from Citrate-Based Loading

The ability of doxorubicin to be accumulated in DPPC/DSPE-PEG2000 (95:5mole ratio) liposomes according to the MnSO₄ and citrate based loadingprocedures was compared. Lipid films were hydrated with 300 mM MnSO₄solution or 300 mM citrate, pH 3.5 and passed through an extrusionapparatus at 55° C. The resulting liposomes were run down a SephadexG-50 column equilibrated with a buffering solution of SHE, pH 7.5 forMnSO₄ containing liposomes and HBS, pH 7.5 for citrate-containingliposomes. After buffer exchange, liposomes were combined withdoxorubicin to give a final drug:lipid weight ratio of about 0.1:1,0.2:1 or 0.3:1. The resulting mixture was incubated at 37° C. for 80minutes. The extent of drug loading was determined as described in themethods by measuring the absorbance at 480 nm to quantify drug; lipidlevels were measured by liquid scintillation counting.

Results summarized in FIG. 7 show that doxorubicin loading efficienciesof >95%, >90% and >80% were achieved in cholesterol-free liposomescontaining MnSO₄ (300 mM) when the initial drug/lipid weight ratios were0.1:1 (panel A), 0.2:1 (panel B) and 0.3:1 (panel C), respectively. Incontrast, cholesterol-free liposomes loaded with doxorubicin accordingto the pH gradient citrate (300 mM citrate, pH 4.0), loading procedureunder the same conditions displayed a substantial reduction inencapsulation efficiency as the doxorubicin/lipid weight ratio wasincreased from 0.1 to 0.3. The latter method could achieve a maximumdrug-to-lipid weight ratio of <0.075. These results demonstrate thatcholesterol-free liposomes can be efficiently loaded with doxorubicin todrug-to-lipid ratios as high as 0.3:1 (w/w) using metal loading whereascitrate-based loading procedures can only achieve a maximumdrug-to-lipid ratios of 0.1:1 (w/w). These data show that metal-basedloading mechanisms are distinct from those relying on maintaining astable pH gradient. Data points represent the mean drug-to-lipid ratioand the error bars represent the standard deviation.

Example 5 Unbuffered Metal Loading Causes Collapse of the TransmembranepH Gradient

The effect of doxorubicin loading on the transmembrane pH gradient ofDMPC/Chol liposomes was compared using citrate and manganese loadingtechniques by measuring pH gradients prior to and subsequent to loadingof drug. DMPC/Chol (55:45 mole ratio) lipid films were hydrated with 300mM citrate buffer, pH 3.5, 300 mM MnSO₄ or 300 mM MnCl₂. The resultingMLVs were subjected to 5 freeze-and thaw cycles (freezing in liquidnitrogen and thawing at 40° C.) followed by extrusion at 40° C. Toexchange the external solutions of the liposomes, samples werefractionated on Sephadex G-50 columns. For liposomes with encapsulatedcitrate, the external buffer was exchanged to HBS and for liposomes withencapsulated MnSO₄ and MnCl₂, the external buffer was exchanged to SHE,pH 7.5. Following buffer exchange, doxorubicin was added at a 0.2:1weight ratio at 60° C. Absorbance at 480 nm following detergentsolubilization was assessed to quantify drug and lipid levels weredetermined by liquid scintillation counting.

Results presented in FIG. 8A show that loading of doxorubicin intoliposomes containing encapsulated citrate (squares) and MnSO₄ (circles)was essentially complete within 5 minutes of incubation. Doxorubicinaccumulation employing MnCl₂ (triangles) was less complete in relationto MnSO₄ and citrate loading. Data points represent the meandrug-to-lipid ratios of at least three replicate experiments and theerror bars indicate the standard deviation.

Transmembrane pH gradients of the formulations before and afterdoxorubicin loading were measured using [14C]-methylamine. Briefly,[14C]-methylamine (0.5 (Ci/mL) was added to the liposome solutionsprepared above. After 15 minutes, 150 (L aliquots were passed down 1 mLSephadex G-50 columns equilibrated in HBS to remove unencapsulatedmethylamine. Lipid and methylamine concentrations before and aftercolumn chromatography were determined by scintillation counting. Thetransmembrane pH gradient was calculated according to the relationship:pH=log{[H+]inside/[H+]outside=log{[methylamine]inside/[methylamine]outside}.

As shown in FIG. 8B, following the establishment of the pH gradient, butprior to doxorubicin loading, the formulations with encapsulated citrate(column 1), MnSO₄ (column 3), MnCl₂ (column 5) exhibited measured pHgradients of 3.4, 1.6 and less than 0.18, respectively. These resultsindicate that transmembrane pH gradients are smaller when manganesesolutions are utilized in relation to citrate. Following addition ofdoxorubicin to liposomes containing encapsulated citrate, the pHgradient decreased from 3.4 (column 1) to 2.3 (column 2). This result isconsistent with previous reports demonstrating doxorubicin-mediatedcollapse of the pH gradient in these formulations. Following doxorubicinloading, the manganese-containing liposomes exhibited no measurable pHgradient (columns 4 and 6) thus demonstrating that these formulationslose their pH gradient during loading of drug. Data points represent themean pH gradient of three separate experiments and the errors barsindicate the standard deviation.

Example 6 Loading Efficiency is Dependent on the Metal Ion Employed

Loading of irinotecan into DSPC/DSPE-PEG2000 (95:5 mole ratio) liposomesencapsulating manganese sulfate or copper sulfate solutions was carriedout in order to compare the loading efficiency of the two differentmetals.

Lipid films were hydrated in a solution of either 300 mM MnSO₄ or 300 mMCuSO₄. The resulting multilamellar vesicles (MLVs) were extruded at 60°C. and the LUVs were buffer exchanged into SHE, pH 7.4. Drug loading wasinitiated by the addition of irinotecan to the resulting solution at a0.1:1 drug-to-lipid weight ratio at 60° C. The extent of drug loadingwas measured as described and absorbance was measured at 370 nm.

Results in FIG. 9 demonstrate that manganese loading of irinotecan wasonly 10% complete at the 30-minute time point, whereas irinotecanloading into copper containing liposomes resulted in greater than 95%loading within 5 minutes. These results illustrate that the loadingproperties of liposomes are highly dependent on the identity of themetal ion.

Example 7 Loading of Drug into Cholesterol-Free Liposomes UsingEncapsulated Manganese, Cobalt and Nickel

Uptake of daunorubicin into cholesterol-free liposomes(DSPC/DSPE-PEG2000) was investigated using internal MnSO₄, CoCl₂ andNiSO₄ solutions at various loading temperatures.

Cholesterol-free (DSPC/DSPE-PEG2000, 95:5 mole ratio) liposomesencapsulating manganese were prepared by hydration of lipid films in 300mM MnSO₄ and extrusion was carried out at 75° C. The samples wereexchanged into HBS using a hand held tangential flow dialysis column.The external buffer contained 1.67 mM EDTA to remove any divalentcations. Daunorubicin was loaded at a drug-to-lipid weight ratio of0.1:1 and loading was carried out at 23° C., 37° C. or 60° C. The extentof drug loading was measured by solubilizing the liposomes in detergentfollowed by measuring the absorbance at 480 nm.

Results in FIG. 10A show that loading of daunorubicin intoDSPC/DSPE-PEG2000 (95:5 mole ratio) MnSO₄ containing liposomes is mostefficient at 60° C. whereas loading at 23° C. and 37° C. occurred to alesser extent. Daunorubicin to lipid ratios (mol:mol) of 0.07 can beachieved when the loading temperature is at 60° C.

Cobalt containing DSPC/DSPE-PEG2000 (95:5 mole ratio) liposomes wereprepared by hydration of lipid films in 150 mM CoCl₂. MLVs were extrudedat 75° C. and the exterior buffer was then exchanged by dialyzingagainst HBS overnight. The liposomes were then further exchanged intoHBS using a hand held tangential flow dialysis column to remove anyresidual CoCl₂. Daunorubicin was loaded at 23, 37 and 60° C. at adrug/lipid weight ratio of 0.1:1. The extent of daunorubicin loading wasdetermined by measuring the absorbance at 480 nm after solubilization ofthe liposomes. Lipid levels were determined by liquid scintillationcounting.

Daunorubicin was efficiently loaded into CoCl₂ containingDSPC/DSPE-PEG2000 (95:5 mole ratio) liposomes at 60° C. (see FIG. 10B).At 60° C., loading resulted in >95% encapsulation of daunorubicin within5 minutes. Loading at 23° C. and 37° C. was less efficient and a 60minute incubation at 37° C. was required to achieve 80% drugencapsulation.

Liposomes containing DSPC/DSPE-PEG2000 (95:5 mole ratio) andencapsulating NiSO4 were prepared as described in the previous examples.Lipid films were hydrated in 300 mM NiSO4 and the external buffer of theliposomes was exchanged by passage through a Sephadex G-50 columnequilibrated with SHE, pH 7.4. Daunorubicin was added such that theinitial (prior to loading) drug-to-lipid weight ratio was 0.2 to 1 andloading was carried out at 60° C. Loading efficiencies of daunorubicinwere measured as described above by UV absorption.

Results in FIG. 10C demonstrate that incubation of daunorubicin withNiSO4 containing DSPC/DSPE-PEG2000 liposomes at 60° C. resulted ingreater than 75% drug encapsulation within 5 minutes.

Example 8 Loading of Drug into Cholesterol-Free Liposomes EmployingEncapsulated Copper

Copper loading of epirubicin into DSPC/DSPE-PEG2000 (95:5 mole ratio)liposomes was also examined.

Copper containing DSPC/DSPE-PEG2000 (95:5 mole ratio) liposomes wereprepared as described in the previous examples. Lipid films werehydrated in 300 mM CuSO₄ and extrusion was carried out at 70° C. Theexternal buffer was replaced with SHE, pH 7.4 by passing liposomesthrough a Sephadex G-50 column equilibrated with SHE buffer prior toloading. Epirubicin was added to the copper-containing liposomes at adrug-to-lipid weight ratio of about 0.2:1 and loading was carried out at60° C. Epirubicin and lipid levels were assayed by spectrophotometry andscintillation counting respectively. To quantify epirubicin, theabsorbance was measured at 480 nm after solubilizing the liposomepreparation with detergent.

FIG. 11 shows that loading of epirubicin into DSPC/DSPE-PEG2000 (95:5mole ratio) liposomes resulted in >95% drug accumulation within 5minutes when uptake occurred at 60° C.

Example 9 Metal Loading of Cholesterol-Containing Liposomes

Uptake of doxorubicin, daunorubicin and topotecan into DSPC/Chol (55:45mole ratio) liposomes was investigated using liposomes prepared toencapsulate copper and cobalt.

DSPC/Chol (55:45 mole ratio) liposomes encapsulating cobalt wereprepared as described above by hydration of lipid films in a solution of300 mM CoCl₂. The external buffer was exchanged by column chromatographyto SHE, pH 7.5. Loading was initiated by the addition of doxorubicin ata drug-to-lipid weight ratio of approximately 0.1:1. Liposomes were thenincubated at 60° C. to facilitate drug loading. The extent of drugloading was measured as described previously by solubilization of thesamples with detergent followed by measurement of the absorbance at 480nm.

Results in FIG. 12A show that within 10 minutes, >90% of the added drugwas encapsulated.

Copper sulfate containing DSPC/Chol (55:45 mole ratio) liposomes wereprepared by hydration of a lipid film in 300 mM CuSO₄. The resultingMLVs were extruded at 70° C. and the external solution was exchanged toHBS by passage through a Sephadex G-50 spin column. The buffer exchangedliposomes were loaded at 60° C. with daunorubicin at a 0.1:1, 0.2:1 or0.4:1 drug-to-lipid weight ratio. Liposomes were solubilized indetergent prior to determining drug levels by measuring the absorbanceat 480 nm.

Results in FIG. 12B indicate that drug loading into DSPC/Chol (55:45mole ratio) liposomes loaded using encapsulated CuSO₄ was efficientwith >90% of the added drugs encapsulated within 5 minutes at 60° C.

DSPC/Chol (55:45 mole ratio) liposomes encapsulating 300 mM CuSO₄ wereprepared as described for FIG. 12B except that the external buffer wasexchanged to SHE, pH 7.4. The liposomes were then incubated withtopotecan at a 0.1:1 drug/lipid weight ratio at 37° C. The extent ofloading was monitored for 2 hours at the indicated time points byquantifying drug absorbance at 380 nm and lipid by liquid scintillationcounting. Drug was quantified by measuring absorbance at 380 nm.

FIG. 12C indicates that the loading of topotecan into DSPC/Chol (55:45mole ratio) liposomes was essentially 100% (>95%) complete within 30minutes.

Example 10 Metal Loading of a Number of Different Drugs into UnbufferedLiposomes Containing Passively Encapsulated Drug

Loading of daunorubicin or irinotecan into various liposomes containinga passively encapsulated drug was investigated under a number ofconditions.

Daunorubicin uptake into cisplatin-containing liposomes was measuredaccording to the following procedures. DSPC/DSPE-PEG2000 (95:5 moleratio) or DMPC/Chol (55:45 mole ratio) liposomes were prepared accordingto the materials and methods of the preceding examples. The lipid filmswere hydrated in 150 mM MnCl₂ or 150 mM CUCl₂, respectively, with 8.5mg/mL cisplatin at 80° C. The MLVs were extruded at 75° C. Precipitatedcisplatin was removed by centrifugation and the samples were thendialyzed against HBS overnight. Samples containing CuCl₂ were furtherexchanged into HBS using a hand held tangential flow dialysis column toremove any residual CuCl₂ or cisplatin. Daunorubicin was loaded intocisplatin/MnCl₂ and cisplatin/CuCl₂ containing liposomes at a drug/lipidweight ratio of 0.1:1 at an incubation temperature of 60° C. The initialcisplatin drug/lipid weight ratio was 0.01:1 for both liposomecompositions. The extent of drug loading was measured as describedpreviously by solubilization of the samples with detergent followed bymeasurement of the absorbance at 480 nm.

FIGS. 13A and 13B show that DSPC/DSPE-PEG2000 (95:5 mole ratio) andDMPC/Chol (55:45 mole ratio) liposomes preloaded with cisplatin can beloaded with a second drug (daunorubicin) when using either manganese- orcopper-based active loading, respectively. Furthermore, daunorubicinencapsulation was not as efficient using MnCl₂ compared to CuCl₂.

Loading of daunorubicin or irinotecan into DPPC/Chol (55:45 mole ratio)liposomes containing either passively entrapped carboplatin orcisplatin, respectively, was analyzed using nickel or copper loading.Lipid films were hydrated in 300 mM NiSO₄ or 75 mM CuCl₂+150 mM CuSO₄with 40 mg/ml carboplatin or 8.5 mg/mL cisplatin, respectively. MLVswere extruded at 70° C. Nickel-containing samples were dialyzedovernight against 1 L 300 mM sucrose, 20 mM HEPES, pH 7.4, while samplescontaining copper were exchanged into SHE, pH 7.4, by chromatography onSepharose columns containing CL4B resin. Daunorubicin was loaded at 37°C. at a drug-to-lipid weight ratio of 0.1:1. Irinotecan was loaded intoliposomes as previously described at 60° C. at a drug-to-lipid weightratio of 0.1. Drug and lipid levels were measured using procedurespreviously described.

Results summarized in FIGS. 13C and 13D illustrate that DPPC/Chol (55:45mole ratio) liposomes prepared with either nickel or copper ionsolutions containing a platinum drug, efficiently load a second drug.

Example 11 Metal Loading Combined with Ionophore-Mediated LoadingTechniques Results in Encapsulation of Multiple Agents

Combining metal loading with an additional active loading mechanismresults in efficient encapsulation of both doxorubicin and vincristineinto a single liposome. Metal loading of doxorubicin followed byionophore-mediated loading of vincristine is detailed below.

DSPC/cholesterol liposomes (55:45 mole ratio) were prepared as describedin the preceding examples except that lipid films were hydrated in 300mM MnSO₄ and the lipid marker 14C-CHE was used. The resulting MLVs wereextruded at 65° C. and then passed through a Sephadex G-50 column thathad been pre-equilibrated with 300 mM sucrose, 20 mM HEPES and 15 mMEDTA (pH 7.5). Doxorubicin was then added in a 0.2:1 drug-to-lipidweight ratio and further incubated at 60° C. for 60 minutes.

Following loading of doxorubicin, the divalent cation ionophore A23187(1? g ionophore/(mol lipid) was added to the liposomes and the mixturewas incubated at room temperature for 3 minutes to facilitate A23187incorporation into the bilayer. Subsequently, vincristine was added tothe mixture and incubated at 50° C. for 100 minutes. A small amount ofradiolabeled vincristine was added to the drug preparation to facilitatedrug quantitation. Drug uptake was performed at a 0.05:1 vincristine tolipid weight ratio. Vincristine and lipid was quantified byscintillation counting following liposome solubilization with detergent.Absorbance at 480 nm was used to quantify doxorubicin levels.

FIG. 14 shows that liposomes preloaded with doxorubicin (circles)through metal loading display near maximum encapsulation ofionophore-mediated loading of vincristine (squares) after 40 minutes ofincubation at 50° C., with no significant leakage of doxorubicin duringvincristine encapsulation. The data points represent the meandrug-to-lipid ratio of three separate experiments and the error barsindicate the standard deviation.

Example 12 Metal Loading of two Drugs in the Absence of IonophoreResults in Efficient Encapsulation of two Drugs

The preceding examples have made use of either passive orionophore-mediated loading procedures in combination with active metalloading to result in encapsulation of two drugs into liposomes ofvarious compositions. The following example demonstrates that metalloading alone can be utilized to actively load two drugs into a singleliposome. Doxorubicin and irinotecan were loaded into DSPC/Cholesterolliposomes as described below.

DSPC/Chol liposomes (55:45 mole ratio) were prepared as detailedpreviously with encapsulated 300 mM CuSO₄. The extruded liposomes werepassed through a Sephadex G-50 column that had been pre-equilibratedwith SHE, pH 7.5. Irinotecan was loaded first at a drug-to-lipid moleratio of 0.2:1 at 60° C. to approximately 100% encapsulation. Followingthis, doxorubicin was incubated at 60° C. at a drug-to-lipid mole ratioof 0.15:1 with the irinotecan-containing liposomal formulation to allowsufficient loading of doxorubicin. Irinotecan levels were measured bymeasuring the absorbance at 370 nm using a standard curve prepared inthe presence of doxorubicin to account for its absorbance at 370 nm.Similarly, doxorubicin concentrations were determined by measuringabsorbance at 480 nm using a standard curve prepared in the presence ofirinotecan to account for its absorbance at 480 nm. As a control,individual uptake of each drug was measured separately into liposomes ofthe same composition.

The results summarized in FIG. 15 illustrate that doxorubicin andirinotecan can be efficiently loaded into a single liposome using theactive metal loading procedure of the invention. The results representthe mean drug-to-lipid ratio of three separate experiments and the errorbars indicate the standard deviation.

Example 13 Drug Release Rates in Vivo are Dependent on the Nature of theMetal ion

The ability of different internal loading mediums to control the releaseof daunorubicin from DSPC/DSPE-PEG2000 (95:5 mole ratio) liposomes invivo, was investigated using 150 mM citrate, pH 4.0, 300 mM CuSO₄ and300 mM MnSO₄. DSPC/DSPE-PEG2000 liposomes were prepared as described andextruded at 75° C. The external solution was exchanged to HBS bydialysis against HBS. Daunorubicin was loaded at a drug-to-lipid weightratio of about 0.1:1 and loading was carried out at 60° C. Daunorubicinloading was measured as described in the preceding examples using EDTAin the solubilization buffer. The drug-loaded liposomes were thenintravenously administered to Balb/c mice at lipid doses of 100 mg/kgand daunorubicin doses of 10 mg/kg. Blood was recovered 24 hours afteradministration by cardiac puncture (3 mice per time point) and collectedinto EDTA-containing tubes. Plasma lipid concentrations were determinedby liquid scintillation counting. Daunorubicin was extracted from plasmaas follows:

A defined volume of plasma was adjusted to 200 (L with distilled waterfollowed by addition of 600 (L of distilled water, 100 (L of 10% SDS and100 (L of 10 mM H₂SO₄. The resulting mixture was mixed and 2 mL of 1:1isopropanol/chloroform was added followed by vortexing. The samples werefrozen at −20° C. overnight or −80° C. for 1 hour to promote proteinaggregation, brought to room temperature, vortexed and centrifuged at3000 rpm for 10 minutes. The bottom organic layer was removed andassayed for fluorescence intensity at 500 nm as the excitationwavelength (2.5 nm bandpass) and 550 nm as an emission wavelength (10 nmbandpass) and using an absorbance wavelength of 480 nm.

FIG. 16 demonstrates that DSPC/DSPE-PEG2000 (95:5 mole ratio) liposomesloaded with daunorubicin employing encapsulated citrate, pH 4.0, CuSO₄and MnSO₄ display differing plasma drug-to-lipid ratios 24 hours afterintravenous administration. These results thus show that drug releasecan be controlled through selection of an appropriate metal ion. Theresults represent the mean drug-to-lipid ratio of at least threeseparate experiments and the error bars indicate the standard deviation.

Example 14 Loading Liposomes in the Presence and Absence of UncomplexedMetal ions

Metal-based loading of drug in the presence and absence of metal ions onthe external surface of phosphatidylglycerol containing liposomes wasexamined and results are depicted in FIGS. 17 and 18.

Liposomes composed of DSPC/DSPG (80:20 mole ratio) were preparedfollowing the procedures as described in Example 1. DSPC and DSPG lipidswere dissolved in chloroform and chloroform/methanol/water (50:10:1v/v), respectively. The lipids were then combined in appropriate amountsfor each formulation. Solvent was removed under a steady stream of N₂gas while maintaining the temperature at 70° C. and put under vacuum for5 minutes. The resulting lipid films were redissolved in chloroform tofurther remove any methanol or water and then solvent was removed asbefore and dried under vacuum to remove any residual solvent. Thesamples were subsequently rehydrated in 150 mM CuSO₄, pH 7.4 (pHadjusted with TEA) and the resulting MLVs were extruded at 70° C.Liposome samples were either run down a 15 mL Chelex-100™ (BioRad)column equilibrated with 150 mM NaCl at 0.5 mL/min or buffer exchangedinto saline and further exchanged into 300 mM sucrose, 20 mM HEPES, pH7.4 using tangential flow. Liposomes that were passed through theChelex-100™ column were subsequently exchanged into 300 mM sucrose, 20mM HEPES, pH 7.4 using tangential flow.

Both liposome preparations were then loaded at 37, 50 and 60° C. withirinotecan at a drug to lipid weight ratio of 0.1:1 as described above.Drug uptake was assayed using liquid scintillation counting to determinelipid concentrations and absorbance at 370 nm to determine irinotecanconcentrations after solubilization in detergent.

Results depicted in FIG. 17 reveal that loading of irinotecan intoDSPC/DSPG (80:20 mole ratio) liposomes was enhanced when the liposomepreparation was passed through a Chelex-100™ column to remove externalmetal ions. In contrast, results shown in FIG. 18 demonstrate thatloading of irinotecan into liposomes exchanged into a solution notcontaining a chelating agent loaded at a reduced rate. Although notwishing to be bound by any particular theory, removal of metal ionsassociated with the negatively charged liposomal surface by complexingthe ions with a chelating agent may reduce metal-drug interactions onthe outer surface of the membrane thereby increasing the amount of freedrug that may cross the membrane to become entrapped in the internalcompartment of the liposome.

Example 15 Methods for the Removal of Metal ions from the ExternalSolution of Liposomes

Copper-based loading of irinotecan into DSPC/DSPG (80:20 mole ratio)liposomes was investigated after removal of Cu²⁺ from the externalsolution using two different techniques both reliant on chelation of theexternal metal. The first technique involved removal of the copper bypassage through a Chelex™ column and the second technique involvedexchanging the liposomes into a buffer containing EDTA.

DSPC/DSPG (80:20 mole ratio) were prepared as in Example 14, except thatsamples were rehydrated in 150 mM copper gluconate, pH 7.4 (pH adjustedwith TEA). External copper was removed by: i) passage through a 15 mLChelex-100 column equilibrated in 300 mM sucrose, 20 mM HEPES, pH 7.4;or ii) by buffer exchange into saline and then into 300 mM sucrose, 20mM HEPES, 30 mM EDTA, pH 7.4 (SHE buffer) using tangential flow.

Both liposome preparations were then loaded at 37, 50 and 60° C. withirinotecan at a drug to lipid weight ratio of 0.1:1 as described inExample 14. Aliquots (100 μL) were removed at various time points afterinitiation of loading and applied to a Sephadex G-50 spin column. Thesamples were then solubilized in detergent and drug and lipidquantitation was performed as previously described in Example 14.

Meta-based loading of irinotecan after removal of external metal bypassage through a chelation column (FIG. 20) is comparable to loadingafter exchanging the liposomes into an EDTA-containing solution (FIG.19). These results thus demonstrate that various means may be employedto remove exterior metal ions from negatively charged membranes withoutconsiderably affecting loading efficiency.

Example 16 Pharmacokinetics of Phosphatidylglycerol-Containing LiposomesCo-Loaded With Daunorubicin and Carboplatin

The retention of daunorubicin and carboplatin co-encapsulated inPG-containing liposomes was investigated by passive loading ofcarboplatin followed by metal loading of daunorubicin.

Daunorubicin and carboplatin were encapsulated in DSPC/DSPG (80:20 moleratio), DSPC/SM/DSPG (75:5:20 mole ratio) and DSPC/SM/DSPG (70:10:20mole ratio) liposomes. The liposomes were prepared following theprocedures as described in preceding examples. DSPG was dissolved in asolution of 50:10:1 chloroform/methanol/water (v/v) and the radioactivemarker 14C-CHE was added to the preparation to quantify lipid. The lipidfilms were rehydrated in 150 mM CuSO₄, 20 mM histidine, pH 7.4containing 80 mg/mL carboplatin (with 4% DMSO to improve carboplatinsolubility). After extrusion, samples were centrifuged to removeunencapsulated carboplatin. Liposomes exchanged into SHE buffer wereloaded with ³H-daunorubicin. Mice were administered liposomes at a doseof 100 mg/kg lipid. Liquid scintillation counting was used to quantitatedaunorubicin and lipid. Plasma carboplatin levels were determined byatomic absorption.

Results in FIG. 21 indicate that dual loaded DSPC/DSPG (80:20 moleratio), DSPC/SM/DSPG (75:5:20 mole ratio) and DSPC/SM/DSPG (70:10:20mole ratio) liposomes display enhanced plasma lipid levels at varioustime points after intravenous administration, although liposomesprepared with 10 mol % sphingomyelin exhibited lower lipid levels inrelation to liposomes with 5 mol % sphingomyelin. The liposomeseffectively altered the pharmacokinetics of the drug as demonstrated bythe high levels daunorubicin and carboplatin remaining in the bloodcompartment after administration (see FIGS. 22 and 23). Liposomesprepared with DSPC/DSPG (80:20 mole ratio) and DSPC/SM/DSPG (75:5:20mole ratio) exhibited the highest daunorubicin and carboplatin levels inrelation to DSPC/SM/DSPG (70:10:20 mole ratio) liposomes.

Although the foregoing invention has been described in some detail byway of illustration and examples for purposes of clarity andunderstanding, it will be readily apparent to those of skill in the artin light of the teachings of this invention that changes andmodification may be made thereto without departing from the spirit ofscope of the appended claims. All patents, patent applications andpublications referred to herein are incorporated herein by reference.

1. A composition comprising liposomes containing an internal solution,the internal solution comprising one or more encapsulated transitionmetal ions and one or more encapsulated therapeutic agents, providedthat if the one or more therapeutic agents is solely doxorubicin, theone or more encapsulated metal ions is not solely manganese, wherein theliposomes do not comprise an ionophore.
 2. The composition of claim 1,wherein the internal solution comprises a metal compatible solution. 3.(canceled)
 4. The composition of claim 1, further comprising in anexternal solution to the liposome.
 5. The composition of claim 4,wherein the external solution and external surfaces of the liposomescontain substantially no uncomplexed metal ions.
 6. The composition ofclaim 5, wherein the external solution comprises a metal chelatingagent.
 7. The composition of claim 4, wherein the external solution andthe internal solution have substantially the same pH.
 8. The compositionof claim 1, wherein the liposomes are of low cholesterol.
 9. Thecomposition of claim 1, wherein the one or more metal ions are firsttransition series metal ions.
 10. The composition of claim 1, whereinthe one or more metal ions are selected from the group consisting of:Fe, Co, Ni, Cu, Zn, V, Ti, Cr, Rh, Ru, Mo, and Pd ions.
 11. Thecomposition of claim 1, wherein the one or more metal ions are selectedfrom the group consisting of: Fe, Co, Cu and Zn ions.
 12. Thecomposition of claim 1, wherein the one or more metal ions are selectedfrom the group consisting of: Zn, Co, and Cu ions.
 13. The compositionof claim 1, wherein the one or more metal ions comprise Cu ions.
 14. Thecomposition of claim 10, wherein the internal solution further comprisesMn ions.
 15. The composition of claim 1, wherein the pH of the internalsolution is in the range of about 6.0 to about 8.5.
 16. The compositionof claim 1, wherein the liposomes comprise one or more lipids that arenegatively charged at physiological pH.
 17. The composition of claim 16,wherein the one or more negatively charged lipids are selected from PGand PI.
 18. The composition of claim 1, wherein the therapeutic agent isan antineoplastic agent. 19-43. (canceled)